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23?and?24 Human colonic HCT-116 cells were grown in 60-mm tissue culture dishes to 70% to 80% confluency. Cells were treated with 10 ��g/mL pepstatin A (MP Pharmaceuticals, Santa Ana, CA) and 10 ��g/mL E-64-d (Peptide Institute, Osaka, Japan) 1 hour before exposure to 1 hour of normoxia or hypoxia [1% O2, hypoxic glove box (Coy Labs, Grass Lake, MI)] and stimulated by 5?��g/mL rapamycin (LC Laboratories). Cells were fixed and permeabilized, as previously described.25 The primary antibody for LC3 (1:200; number 4108; Cell Signaling, Danvers, MA) was used according to the manufacturer's specifications, followed by fluorescein isothiocyanate�Cconjugated goat anti-rabbit secondary antibody (1:1000; number A-11008; Invitrogen). Images were acquired using a Zeiss 710 microscope and Zeiss Zen software version 2009 (Carl Zeiss, Thornwood, NY). Then, puncta (green fluorescent protein) and cells (DAPI; number Oxalosuccinic acid H-1200; Vector Laboratories, Burlingame, CA) were counted using ImageJ software version 1.48u4 (Wayne. S. Rasband, NIH, Bethesda, MD). WT healthy and injured ileal tissues were deparaffinized using ethanol and xylenes. Antigen retrieval was performed by using citrate buffer (pH 6) in a pressure cooker. Primary LC3 antibody (1:200; number 4108; Cell Signaling, Danvers, MA) was incubated overnight according to manufacturer's specifications, followed by Alexa Fluor 647 goat anti-rabbit secondary antibody (1:1000; Invitrogen). Selleck Bosutinib DAPI (number H-1200; Vector Laboratories, Burlingame, CA) was used to counterstain the sections. Images were acquired by a Leica SPA microscope (Leica Microsystems Inc., Buffalo Grove, IL). Analysis of LC3 puncta was performed using ImageJ software, generating a basal cutoff of LC3 puncta (threshold, 10), then using the Analyze Particle feature for counting to generate a mean of positive pixels per cell (%) �� SEM of three fields of view Selleckchem Vandetanib per sample.26 Immunofluorescence experiments were performed using this secondary antibody instead of the one previously mentioned, because of the change in confocal microscope. Unless specifically noted, statistical analyses were performed using GraphPad Prism version 5.0a (GraphPad, La Jolla, CA). Comparisons of mouse studies were made with nonparametric analysis of variance and then a U-test. All graphs depict means?��?SEM. Experiments were considered statistically significant if P?